The large Gunnera’s (G. tinctoria and G. manicata) in Europe in relation to EU regulation 1143/2014

Incorrect labelling of plants in the horticultural trade and misidentification is widespread. For the inspection services of the EU member states, correct identification of G. tinctoria has become important since the species was added to the List of Union concern in accordance with EU regulation 1143/2014 in August 2017. In the horticultural trade Gunnera plants are generally of modest dimensions and rarely flowering, so that the major distinguishing morphological characters for the identification of the two large species, G. tinctoria and G. manicata, are missing. As G. tinctoria is included in the EU regulation, its trade is prohibited, although the closely related species, G. manicata is not included on the list. Given that it is often difficult to distinguish between these two large herbaceous species using morphological attributes we used standard chloroplast DNA barcode markers, supplemented at a later stage by ITS markers. Plant material of putative G. tinctoria or G. manicata was obtained from the native and introduced range, both from “wild” sources, botanical gardens, and the horticultural trade. In western Europe plants circulating in the horticultural trade turned out to be predominantly G. tinctoria, with only one plant in cultivation identified as true G. manicata and the G. manicata found in botanical gardens was a hybrid recently described as G. x cryptica.


Introduction
Incorrect labelling of plants in the horticultural trade and misidentification is widespread and may be caused by negligence or wilful disrespect of regulations [1][2][3][4][5]. Mislabelling may consist of simple misspelling of names or considering a variety as a true species, using synonyms or just preferring a name that sounds nice or a name that customers are familiar with. The latter two cases are indeed considered intentional misidentification of the plant in the horticultural trade. If mislabelled species are not regulated or do not pose a threat to the natural environment, only the customer should be affected (e.g. by not having the plant that they wished for). However, if the plant species is a potential threat to the environment or is regulated, the problem may be much more serious [6]. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Gunnera L. is the single genus in the family Gunneraceae. According to Plants of the World online it comprises 63 species. Other authors sometimes mention it to be represented by 30-40 species [e.g. 7], mostly distributed in the Southern Hemisphere. The subgenus Panke to which the large perennial species found in European gardens belong comprises some 20 species found in Central and South America and Hawaii. The species of subgenus Panke are unique in having large, triangular scales between the leaves on the rhizomes and are characterised by rosettes of huge palmate leaves that account for their aesthetic value [8].
Despite questions about the nomenclature for the proper naming of Gunnera manicata Linden ex André and G. tinctoria (Molina) Mirb [9][10][11], the common opinion is that two kinds of large Gunnera's predominate in western Europe. One of them is an invasive species and included in EU regulation 1143/2014 and named Gunnera tinctoria [12]. In Europe this species can form extensive long-lived naturalised populations under the high rainfall, high humidity conditions found in parts of Ireland, Portugal and the British Isles, which rarely experience extreme low temperatures [12]. Outside of Europe naturalised populations can be found under similar environmental conditions in the United States and in New Zealand, where invasive populations can also be found [13].
In the horticultural trade the typical Gunnera plants used are of modest dimensions and rarely flowering, so that the major distinguishing characters that might be used to distinguish between the two species are missing. It is also clear that it is not easy to distinguish between G. tinctoria and G. manicata based on morphological attributes alone [9,12]. As G. tinctoria is included in the EU regulation, the trade of this species is prohibited, necessitating the use of other than macromorphological characteristics to clearly distinguish between the two species. The approach used in this study was to use standard chloroplast DNA barcode markers supplemented later with ITS markers to successfully distinguish between the two species.

Plant material
The plant material used in this study is shown in Table 1 A sample from the native range in Chile was obtained in January 2020 (Vasquez-Garcia s.n. silica dried).
Additional measurement on leaves and inflorescence from Gunnera plants in the Hortus Botanicus Leiden were made in July 2022, to match molecular findings with morphological features. For each plant 5 leaves and 4 inflorescences, 10 branches of each inflorescence at midsection of the inflorescence, were used (1 G. tinctoria, 2 G. "manicata").
Additional information regarding the ethical, cultural, and scientific considerations specific to inclusivity in global research is included in the S1 File.

DNA extractions
Approximately 1 gram of plant material was ground in 5 ml GH+ grinding buffer (6 M guanidine hydrochloride, 0.2 M sodium acetate pH 5.2, 25 mM EDTA, and 2.5% PVP-10). Genomic DNA was isolated from 75 μl of the homogenate with the DNeasy plant mini kit (Qiagen, Venlo, The Netherlands) using 50 μL prewarmed (65˚C) AE buffer for elution. DNA was stored at −20˚C until used.

PCR analyses
The PCR reactions for the chloroplast rbcL gene, the trnH-psbA intergenic spacer and nuclear ITS (partial 18S, ITS1, 5.8S, ITS2, partial 28S) loci were performed in 25 μL reaction mixes containing 200 nM of either primers rbcL-a F and rbcLa SI_Rev, trnH2 and psbAF or ITS5 and ITS4 (Table 2) respectively, 1 x MyFiTM Mix (Bio-line, Taunton, USA) and 2 ul genomic DNA. The cycle conditions for rbcL and trnH-psbA loci were as follows: 5 min at 95˚C, followed by 5 cycles of 30 s at 94˚C, 30 s at 45˚C, 30 s at 72˚C and 35 cycles of 30 s at 94˚C, 30 s at 50˚C, 30 s at 72˚C and a final extension for 10 min at 72˚C. The cycle condition for the ITS locus was as follows: 5 min at 95˚C, followed by 40 cycles of 30 s at 94˚C, 30 s at 52˚C, 100 s at 72˚C and a final extension for 10 m in at 72˚C.

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The large Gunnera's (G. tinctoria and G. manicata) in Europe

Sanger sequencing
Two μl of ExoSAP-IT Express (Thermo Fisher Scientific, Bleiswijk, the Netherlands) was added to 5 μl PCR product and incubated (4 min 37˚C, 1 min 80˚C, 1 min 20˚C) preceding bidirectional cycle sequencing with the BigDye Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Bleiswijk, the Netherlands) using amplification primers as sequencing primers in separate reactions according to the manufacturer's instructions. Cycle sequence products were purified with the DyeEx 2.0 Spin Kit (Qiagen,Venlo, the Netherlands) and sequenced using a 3500 Genetic Analyzer (Thermo Fisher Scientific, Bleiswijk, the Netherlands). Consensus sequences were generated from an assembly with trace files from both Sanger sequencing runs in Geneious Prime (Biomatters Auckland, New Zealand). Amplification primer sequences were trimmed in the assembly and, when needed, additional trimming was performed to obtain high-quality (PHRED > 30) consensus sequences.

Morphological characteristics of plants in cultivation
The perceived morphological distinction between the two Gunnera species encountered in western Europe, as described in detail by Shaw et al. [19], was examined using measurements on plants in the Hortus Botanicus at Leiden, that were also sampled for our molecular work ( Table 3). The inflorescence in G. tinctoria is of more modest dimensions, based on total length, length of the peduncle and diameter. Likewise, the leaves are of more modest dimensions. The partial inflorescences in G. tinctoria are shorter but have a larger diameter. These findings are in line with the comparative table reported in Shaw et al. [19] Concerning the nomenclature issue for the proper naming of G. manicata and the resulting hybrid with G. tinctoria we refer to Shaw et al. [19]. The plants in cultivation in western Europe and found in New Zealand that are considered to be G. manicata are in fact hybrids and should be named G. x cryptica J.M.H.Shaw.
The Gunnera plants in the horticultural trade in Belgium were consistently mislabelled as G. manicata, whereas images of the flowering plants clearly identified them as G. tinctoria [5]. Likewise, Gunnera plants in the horticultural trade in the Netherlands are likely to have been mislabelled either accidentally or on purpose.

Molecular characterisation of the plants in cultivation
The putative G. manicata and G. tinctoria material collected from the Hortus Botanicus Leiden had identical rbcL sequences and differed only 1 nt in their trnH-psbA spacer sequence (Table 4). Blast searches with the trnh-psbA spacer sequence at NCBI GenBank showed that there was 99.7% identity overlap with a G. chilensis Lam. (synonym of G. tinctoria) accession (AB250752), with no G. manicata trnH-psbA sequences present in NCBI GenBank at the time.

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The large Gunnera's (G. tinctoria and G. manicata) in Europe As the inter-specific variation of the trnH-psbA loci is often large enough for species identification, it was assumed that the specimen labelled as G. manicata from the Hortus Botanicus Leiden was also G. tinctoria. The G. tinctoria plants collected in New Zealand contained similar cpDNA markers. However, plants in the horticultural trade sampled in July 2018 did give contrasting results as a plant resembling a depauperate G. manicata (Valkenburg 3948) was identified as G. tinctoria based on their cpDNA.
In addition to the cpDNA markers we also included ITS for our analysis as Bacon et al. [20] has published several ITS sequences for Gunnera species, which show more variation than the use of trnH-psbA markers. To obtain 'true' G. manicata we contacted Stockholm University to verify the presence of living material of the herbarium voucher from the Wanntorp 560 collection [11]. Unfortunately, the herbarium specimen could not be retrieved, nor could any living  . tinctoria (1), and two plants of "G. manicata" (2, 3). Each plant 5 leaves and 4 inflorescences, 10 branches of each inflorescence at midsection of inflorescence.
material that was specifically labelled as Wanntorp 560 G. manicata. However, plants labelled as G. manicata" Klon B" were present in the Department's greenhouse collection. This material (Valkenburg 4433) gave a 100% match with the GenBank data for Wanntrop 560 (trnH-psbA (MH017175) [20] and ITS (AF447740) [11]) and a collection from Brazil (Pouso Redondo, Serra do Matador, 28 Dec 2000),A. Reis s.n. (ITS AF447741 [11]). Young plants transferred from Stockholm to the greenhouse at Wageningen exhibited poor growth and died after six months. All previous samples were then reanalysed and new samples from botanical gardens in Belgium, France and the Azores, as well as wild material collected from Chile and the Azores were included. Of the plants labelled as G. manicata all were found to be hybrids between G. manicata and G. tinctoria with G. manicata being the mother plant, as ambiguous nucleotides were found at the position where the ITS sequences of G. manicata and G tinctoria differ, while the trnH-psbA sequence was identical to that of G. manicata (see Tables 1 and 4). All plants labelled as G. tinctoria from gardens in the Netherlands or material obtained from naturalised or native provenances in Ireland, the Azores, New Zealand and Chile were 100% pure G. tinctoria.
A plant labelled G. manicata that was identified from the horticultural trade (Valkenburg 3948), which had a slightly more condensed inflorescence, was also found to be of hybrid origin but with G. tinctoria as the mother plant. Another plant labelled as G. manicata (Valkenburg 4432) was simply mislabelled and turned out to be pure G. tinctoria.
Clearly, the earlier statement that G. tinctoria does not form viable hybrids [12] is incorrect. An earlier report by Palkovic [21] also reported the occurrence of hybrids between two large Gunnera species, G. insignis (Oerst.) Oerst. and G. talamancana H.Weber & L.E.Mora in Costa Rica, indicating that hybridisation within the genus may be more common than once thought.
Aside from the common problem of misidentification of plant material this study also raises some interesting questions about the identification and origin of the hybrid material. If all the material in gardens or circulating in the horticultural trade that is labelled as G. manicata are hybrids, where did this material come from and when did the hybridisation occur? Given that our results indicate that all naturalised plants are pure G. tinctoria, were any 'pure' G. manicata plants ever introduced?
The status of hybrids in the context of the EU regulation 1143/2014 and the associated delegated act is somewhat ambiguous. Whereas, the EU regulation 1143/2014 defines an alien species under article 3(1) as "any live specimen of a species, subspecies or lower taxon of animals, plants, fungi or micro-organisms introduced outside its natural range; it includes any part, gametes, seeds, eggs or propagules of such species, as well as any hybrids, varieties or breeds that might survive and subsequently reproduce;" The associated delegated act clearly specifies under article 5 (1)(a)(2) "It shall be clearly stated if the risk assessment covers more than one species, or if it excludes or only includes certain subspecies, lower taxa, hybrids, varieties or breeds (and if so, which subspecies, lower taxa, hybrids, varieties or breeds). Any such choice must be properly justified." For the G. tinctoria hybrids, there is no evidence that these are invasive and persist outside of cultivation, as all the naturalised material was identified as G. tinctoria. Based on this information the legislation may need to be revised to account for scenarios where hybrids are less problematic than their parents.

Conclusion
Based on the evidence from this study all plants found in gardens in the Netherlands, Belgium, France and New Zealand and labelled as G. manicata are likely of hybrid origin with G. manicata as the mother plant. In the horticultural trade most plants labelled as G. manicata, are likewise hybrids that have G. manicata as the mother plant. However, some of the plants in the horticultural trade that are labelled as G. manicata and have a slightly more condensed inflorescence, may have G. tinctoria as the mother plant. Confirmation of the extent of hybridisation and the parental contributions will, however, require a more extensive study using a wider range of genotypes. Clearly, several other G. tinctoria plants, including naturalised material, have simply been mislabelled as G. manicata. All plants labelled as G. tinctoria and found either in gardens or as naturalised provenances are 100% pure G. tinctoria.